The invention is disclosed herein in terms of the exemplary preparation of mono-glycosylated influenza viruses. It is contemplated, that the same methods can be used to prepare other viruses with simplified glycan structure including retroviruses such as the human immunodeficiency virus (HIV) and flaviviridae such as Dengue virus, West Nile virus, hepatitis C virus (HCV) and the like.
Influenza is caused by an RNA virus of the orthomyxoviridae family. There are three types of these viruses and they cause three different types of influenza: type A, B and C. Influenza virus type A viruses infect mammals (humans, pigs, ferrets, horses) and birds. This is very important to mankind, as this is the type of virus that has caused worldwide pandemics. Influenza virus type B (also known simply as influenza B) infects only humans. It occasionally causes local outbreaks of flu. Influenza C viruses also infect only humans. They infect most people when they are young and rarely cause serious illness.
Influenza A viruses infect a wide variety of mammals, including man, horses, pigs, ferrets and birds. The main human pathogen, associated with epidemics and pandemics. There are at least 15 known hemagglutinin (H) serotypes and 9 known neuraminidase (N) serotypes. Pigs and birds are believed to be particularly important reservoirs, generating pools of genetically and antigenically diverse viruses which get transferred back to the human population via close contact between humans and animals. Influenza B viruses infect mammals only and cause disease, but generally not as severe as A types. Unlike influenza A viruses, influenza B viruses do not have distinguishable serotypes. Influenza C viruses also infect mammals only, but rarely cause disease. They are genetically and morphologically distinct from A and B types.
There are 4 antigens present in the influenza virus, the hemagglutinin (HA), neuraminidase (NA), nucleocapsid (NA), the matrix (M) and the nucleocapsid proteins (NP). The NP is a type-specific antigen which occurs in 3 forms, A, B and C, which provides the basis for the classification of human influenza viruses. The matrix protein (M protein) surrounds the nucleocapsid and makes up 35-45% of the particle mass. Two surface glycoproteins are seen on the surface as rod-shaped projections. The hemagglutinin (HA) is initially synthesized as a trimeric precursor (HA0) containing three identical protein chains, each of which is proteolytically processed into two subunits, HA1 and HA2, that are held together covalently by a single disulfide bond. HA mediates the attachment of the virus to the cellular receptor. Neuraminidase (NA) molecules are present in lesser quantities in the envelope. Circulating human strains are notorious for their tendency to accumulate mutations from one year to the next and cause recurrent epidemics.
In eukaryotes, sugar residues are commonly linked to four different amino acid residues. These amino acid residues are classified as O-linked (serine, threonine, and hydroxylysine) and N-linked (asparagine). The O-linked sugars are synthesized in the Golgi or rough Endoplasmic Reticulum (ER) from nucleotide sugars. The N-linked sugars are synthesized from a common precursor, and subsequently processed. It is known that addition of N-linked carbohydrate chains is important for stabilization of folding, prevention of degradation in the endoplasmic reticulum, oligomerization, biological activity, and transport of glycoproteins. The addition of N-linked oligosaccharides to specific Asn residues plays an important role in regulating the activity, stability or antigenicity of mature proteins of viruses (Opdenakker G. et al FASEB Journal 7, 1330-1337 1993). It has also been suggested that N-linked glycosylation is required for folding, transport, cell surface expression, secretion of glycoproteins (Helenius, A., Molecular Biology of the Cell 5, 253-265 1994), protection from proteolytic degradation and enhancement of glycoprotein solubility (Doms et al., Virology 193, 545-562 1993). Viral surface glycoproteins are not only required for correct protein folding, but also provide protection against neutralizing antibodies as a “glycan shield.” As a result, strong host-specific selection is frequently associated with codon positions of potential glycosylation. Consequently N-linked glycosylation sites tend to be conserved across strains and clades.
Outbreaks of influenza A virus continue to cause widespread morbidity and mortality worldwide. In the United States alone, an estimated 5 to 20% of the population is infected by influenza A virus annually, causing approximately 200,000 hospitalizations and 36,000 deaths. The establishment of comprehensive vaccination policies has been an effective measure to limit influenza morbidity. However, the frequent genetic drifting of the virus requires yearly reformulation of the vaccine, potentially leading to a mismatch between the viral strain present in the vaccine and that circulating. Thus, antiviral therapies against influenza virus are important tools to limit both disease severity as well as transmission.
The highly pathogenic H5N1 influenza viruses have caused outbreaks in poultry and wild birds since 2003 (Li K S et al. (2004) Nature 430:209-213). As of February 2010, these viruses have infected not only avian species but also over 478 humans, of which 286 cases proved to be fatal (www_who_int/csr/disease/avian_influenza/country/cases_table_2010_02_17/en/index html). The highly pathogenic H5N1 and the 2009 swine-origin influenza A (H1N1) viruses have caused global outbreaks and raised a great concern that further changes in the viruses may occur to bring about a deadly pandemic (Garten R J, et al. (2009) Science 325: 197-201, Neumann G, et al. (2009) Nature 459:931-939). There is great concern that an influenza virus would acquire the ability to spread efficiently between humans, thereby becoming a pandemic threat. An influenza vaccine must, therefore, be an integral part of any pandemic preparedness plan.
Important contributions to the understanding of influenza infections have come from the studies on hemagglutinin (HA), a viral coat glycoprotein that binds to specific sialylated glycan receptors in the respiratory tract, allowing the virus to enter the cell (Kuiken T, et al. (2006) Science 312:394-397; Maines T R, et al. (2009) Science 325:484-487; Skehel J J, Wiley D C (2000) Ann Rev Biochem 69:531-569; van Riel D, et al. (2006) Science 312:399-399). To cross the species barrier and infect the human population, avian HA must change its receptor-binding preference from a terminally sialylated glycan that contains α2,3 (avian)-linked to α2,6 (human)-linked sialic acid motifs (Connor R J, et al. (1994) Virology 205:17-23), and this switch could occur through only two mutations, as in the 1918 pandemic (Tumpey T M, et al. (2007) Science 315:655-659). Therefore, understanding the factors that affect influenza binding to glycan receptors is critical for developing methods to control any future crossover influenza strains that have pandemic potential.
The influenza virus hemagglutinin (HA) is a homotrimeric transmembrane protein with an ectodomain composed of a globular head and a stem region (Kuiken T, et al. (2006) Science 312:394-397). Both regions carry N-linked oligosaccharides (Keil W, et al. (1985) EMBO J 4:2711-2720), which affect the functional properties of HA (Chen T Y, et al. (2008) Vaccine 26:361-371; Ohuchi R, et al. (1997) J Virol 71:3719-3725). HA is the virion surface glycoprotein that attaches the virus to its receptors on host cells and fuses the viral envelope with the membranes of endocytic vesicles to initiate the infectious process. (Crecelius D. M., et al. (1984) Virology 139, 164-177). It is also the virion component that stimulates the formation of protective antibodies. The nature and extent of glycosylation of the HA have been implicated in altering its receptor binding properties and in the emergence of viral variants with enhanced cytopathogenicity (Aytay S., Schulze I. T. (1991) J. Virol. 65, 3022-3028) and virulence (Deshpande K. L., et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 36-40) and in masking its antigenic sites (Skehel J. J., et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1779-1783). Mutational deletion of HA glycosylation sites can affect viral receptor binding (Gunther I, et al. (1993) Virus Res 27:147-160).
The amino acid sequence of the HA and hence the location of its N-glycosylation sites is determined by the viral genome. The structures of these oligosaccharides appear to be determined by their position on the HA (Keil W., et al. (1985) EMBO J. 4, 2711-2710) and by the biosynthetic and trimming enzymes provided by the host cell in which the virus is grown. The plasticity of the viral genome and the host-specified glycosylation machinery can, together, create virus populations that are more heterogeneous in structure and function than could be developed by either process alone. This diversity is considered to be responsible for survival of these viruses and for their ability to overcome the inhibitory effects of neutralizing antibodies and antiviral agents.
The HA appears to have regions that must be glycosylated, others that must be free of oligosaccharides, and still others in which glycosylation may be either advantageous or detrimental to the survival of the virus. Glycosylation sites at certain positions on the HA of influenza A viruses isolated from various animals and humans are highly conserved and therefore appear to be essential for the formation and/or maintenance of functional HA (Gallagher P. J., et al. (1992) J. Virol. 66, 7136-7145). Conversely, the generation of glycosylation sites in certain regions of the HA reduces its transport to the cell surface, and its stability and/or function (Gallagher P., et al. (1988) J. Cell Biol. 107, 2059-2073). It is in the regions in which glycosylation is neither prohibited nor required for the formation of functional HA that oligosaccharide diversity may have a major selective effect, depending on the specific environment in which the virus is expected to grow.
Changes in the peptide sequence at or near glycosylation sites may alter HA's 3D structure, and thus receptor-binding specificity and affinity. Indeed, HAs from different H5N1 subtypes have different glycan-binding patterns (Stevens J, et al. (2008) J. Mol Biol 381:1382-1394).
Mutagenesis of glycosylation sites on H1 and H3 has been studied in the whole-viral system (Chandrasekaran A, et al: (2008) Nat Biotechnol 26:107-113; Deom C M, et al. (1986) Proc. Natl Acad Sci USA 83:3771-3775). Changes in glycosylation could affect receptor-binding specificity and affinity, especially with regard to the most pathogenic H5N1 HA.
Less highly glycosylated or non-glycosylated regions of hemagglutinin continue to mutate to escape from the host immune system. Vaccine design using monoglycosylated HA is disclosed in US Pat. App. Pub. No. 2010/0247571 (Wong et al.). Thus, there is a need for novel and efficient methods for production of monoglycosylated influenza virus particles.